The long-term objective of this proposal is to define mononuclear cells in human muscle that hold promise for optimization of cell-based therapy for muscular dystrophy and myopathy. Recent findings from our laboratory have indicated that specific cell populations in human skeletal muscle may interact with one another, regulating proliferation and differentiation of muscle precursor cells. Using various methods of purification and by working closely with Project 1, which will perform parallel studies on muscle progenitors in zebrafish, we will fractionate several subsets of human muscle precursor cells. These isolated fractions will be tested in both in vitro and in vivo models of skeletal muscle repair using three separate paradigms of myopathy: dystrophin, dysferlin and myotubularin deficiency. The overall goal of this application is that by better defining the mononuclear cells in human skeletal muscle and their interactions with one another, it will be possible to identify a highly proliferative, myogenic cell fraction that is amenable to therapeutic applications. This goal will be achieved via the following specific Aims: Aim 1. Fractionate mononuclear cell populations in human fetal and adult skeletal muscles, study their cell surface similarities, proliferative capacity and myogenic potential in vitro. Aim 2. Use mRNA expression arrays to characterize human myogenic progenitors with high proliferative and repair capacity. Aim 3. Assay the capacity of fractionated human muscle progenitor cells to repair mutant muscle cells in vitro. Aim 4. Determine the restorative and myogenic potential of distinct mononuclear cell populations within human muscle in vivo. The hope is that by working in close synergy with Project 1, we will be able to understand the signals that control human muscle cell proliferation, myogenic commitment and ability to repair diseased muscle. By pursuing each one of these efforts, we will define the most effective cell population(s) in human muscle and demonstrate their effectiveness in pre-clinical mouse models of muscular dystrophy and myopathy. If successful, these findings are likely to make a significant step forward in the field of cell-based therapy for muscular dystrophy.